Post on 12-Mar-2019
PURITY DETERMINATION
OF ADENOVIRUSES
Febri Annuryanti
Supervision:
Dr. Marta Germano Crucell Holland B.V.
Dr. Wim Th. Kok University of Amsterdam
Faculty of Science
University of Amsterdam
July 2013
MSc Chemistry
Analytical Sciences
Master Thesis
PURITY DETERMINATION OF
ADENOVIRUSES
by
Febri Annuryanti
July 2013
Supervisor:
Dr. Wim Th. Kok
Daily Supervisor :
Marta Germano, PhD
DEPARTMENT OF ANALYTICAL DEVELOPMENT
CRUCELL HOLLAND B.V.
Purity determination of adenoviruses
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ABSTRACT
Adenoviruses are potentially useful vectors for vaccination. Crucell
manufactures recombinant adenoviruses on a PER.C6® cell substrate. An extensive
purification is done during the production of recombinant adenoviruses in order to
obtain the pure final products. However, product-related impurities, such as virus
aggregates and virus incomplete particles, can be present in the final product and
may lead to adverse effects or lack of efficacy. Incomplete particles and complete
particles can be separated by cesium chloride (CsCl) density gradient
centrifugation. In addition, a qualitative method to determine the presence of
incomplete particles is available by differential centrifugal sedimentation (DCS).
The goal of this study was to obtain a suitable analytical method for
determining the purity of adenovirus based on the content of product-related
impurities. SEC-HPLC, IEX-HPLC, CE and RP-HPLC were used to distinguish between
the complete and incomplete particles. No separation was observed between the
complete and incomplete particles with SEC-HPLC, IEX-HPLC, and CE techniques.
These results indicated that the complete and incomplete particles have similar
hydrodynamic volume and charged surface. Only RP-HPLC was able to differentiate
between the complete and incomplete particles based on the protein profiles.
Protein 13 and protein 10 were chosen as markers of purity. Protein 13 is
associated with the encapsidation of the viral DNA and protein 10 is universally
present in all forms of adenovirus (both complete and incomplete particles). A
linear correlation was obtained by plotting the percentage of upper band from CsCl
gradient against the ratio of molar concentrations of protein 13 and protein 10.
The percentage of incomplete particles in an adenovirus reference material was
between 4% and 8%.
In conclusion, the purity of adenovirus (with respect to product-related
impurities) could be determined by RP-HPLC. The RP-HPLC method can potentially
replace the DCS method for determining the purity of the adenovirus.
Purity determination of adenoviruses
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PREFACE
This research project is part of a Master in Chemistry. The research was carried out
at Crucell Holland B.V. for 8 months. The objective of the research is exploring new
approaches to determine purity of adenovirus and identifying the product-related
impurities, which are important parts of product characterization.
ACKNOWLEDGEMENTS
I would like to thank to my supervisor Marta Germano, Angel Huidobro and Ewoud
van Tricht (Crucell Holland B.V) and Wim Th. Kok (University of Amsterdam) for
their supervision.
Thanks to all my Crucell colleagues who trained and helped me during my
internship. I wish them big success with their life and career.
Last but not least, thanks to my family for supporting me during my study.
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ABBREVIATIONS
Ad : Adenovirus Ad35WT : Adenovirus type 35 Wild Type AEX : Anion Exchange Chromatography AUC : Analytical Ultracentrifugation BGE : Background electrolyte CE : Capillary Electrophoresis CEC : Capillary Electrochromatography CGE : Capillary Gel Electrophoresis CIEF : Capillary Isoelectric Focusing CITP : Capillary Isotacophoresis CsCl : Cesium Chloride CZE : Capillary Zone Electrophoresis d : Detector pathlength DCS : Differential Centrifugal Sedimentation DNA : Deoxyribonucleic acid DSP : Down Streaming Process EOF : Electroosmotic Flow f : Flow rate GMP : Good Manufacturing Practices HPLC : High Performance Liquid Chromatography ID : Internal diameter of capillary IEX : Ion Exchange Chromatography KDa : Kilo Dalton MDa : Mega Dalton MEKC : Micellar Electrokinetic Capillary Chromatography MS : Mass Spectrometry PDA : Photo Diode Array PVA : Polyvinyl alcohol q : Eluted quantity RfA : Peak area response factor Rfh : Peak height response factor RP-HPLC : Reversed Phase – High Performance Liquid Chromatography RSD : Relative standard deviation SEC : Size Exclusion Chromatography TFA : Trifluoroacetic Acid TRIS : Tris(hydroxymethyl)aminomethane UV / VIS : Ultraviolet / Visible
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TABLE OF CONTENTS
ABSTRACT ....................................................................................................................................... 3
PREFACE ......................................................................................................................................... 4
ACKNOWLEDGEMENTS ................................................................................................................... 4
ABBREVIATIONS ............................................................................................................................. 5
TABLE OF CONTENTS ....................................................................................................................... 6
INTRODUCTION .............................................................................................................................. 8
THEORY......................................................................................................................................... 10
2.1 ADENOVIRUS ......................................................................................................................... 10
2.2 METHOD FOR ANALYSIS OF ADENOVIRUS PURITY AND PRODUCT-RELATED IMPURITIES ........................ 12
2.2.1 UV absorbance .............................................................................................................. 12
2.2.2 Ultracentrifugation ........................................................................................................ 12
2.2.3 Differential Centrifugal Sedimentation (DCS) ................................................................ 13
2.2.4 High Performance Liquid Chromatography (HPLC) ....................................................... 14
2.2.4.1 Size Exclusion Chromatography (SEC-HPLC) ....................................................................... 15
2.2.4.2 Ion Exchange Chromatography (IEX-HPLC) ........................................................................ 16
2.2.4.3 Reversed Phase HPLC (RP-HPLC) ........................................................................................ 17
2.2.5 Capillary Electrophoresis (CE) ........................................................................................ 18
EXPERIMENTAL SECTION .............................................................................................................. 21
3.1 STANDARD AND SAMPLES ......................................................................................................... 21
3.2 DIFFERENTIAL CENTRIFUGAL SEDIMENTATION .............................................................................. 21
3.3 SIZE EXCLUSION CHROMATOGRAPHY .......................................................................................... 21
3.3.1 Materials and Methods ................................................................................................. 21
3.3.2 Preparation of Adenovirus Control Sample and CsCl Fractions ..................................... 21
3.4 ANION EXCHANGE CHROMATOGRAPHY ....................................................................................... 22
3.4.1 Materials and Methods ................................................................................................. 22
3.4.2 Preparation of Adenovirus Control Sample and CsCl Fractions ..................................... 22
3.5 CAPILLARY ELECTROPHORESIS ................................................................................................... 22
3.5.1 Materials and Methods ................................................................................................. 22
3.5.2 Preparation of Adenovirus Control Sample and CsCl Fractions ..................................... 22
3.6 ADENOVIRUS PURITY DETERMINATION USING REVERSED-PHASE HPLC ............................................ 22
3.6.1 Materials and Methods ................................................................................................. 22
3.6.2 Preparation of Adenovirus Control samples and CsCl Fractions .................................... 23
RESULTS AND DISCUSSION ............................................................................................................ 24
4.1 DIFFERENTIAL CENTRIFUGAL SEDIMENTATION (DCS) AND AUC ...................................................... 24
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4.2 SIZE EXCLUSION CHROMATOGRAPHY (SEC-HPLC) RESULT ............................................................. 25
4.3 ANION-EXCHANGE CHROMATOGRAPHY (AEX-HPLC) RESULT ........................................................ 29
4.4 CAPILLARY ELECTROPHORESIS (CE) RESULT ................................................................................. 30
4.4.1 Capillary 1 ...................................................................................................................... 32
4.4.2 Capillary 2 ...................................................................................................................... 34
4.4.3 Capillary 3 with dynamic coating .................................................................................. 34
4.5 REVERSED-PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (RP-HPLC) ............................... 36
CONCLUSION ................................................................................................................................ 43
FUTURE PERSPECTIVE ................................................................................................................... 45
REFERENCES .................................................................................................................................. 46
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INTRODUCTION
Crucell is a global pharmaceutical company that focuses on research,
development, and production of viruses and antibodies used in vaccines against
infectious diseases worldwide. Several products in development are based on
AdVac® technology, in which rare adenoviruses, such as Ad35 and Ad26, are used
as vectors for recombinant vaccines. Adenoviral vectors have been widely
investigated for clinical applications due to their ability to penetrate into the cell
[1-3]. AdVac® vaccination vectors can be manufactured to high titers on a PER.C6®
cell substrate, which does not allow replication-competent adenovirus to form
during the production process. These vectors combine all the advantages of
adenoviral vectors commonly used for vaccination, such as high production yields,
strong immunogenicity, and excellent safety profile, together with accurate dose
control of the vector-based vaccine. Thus, AdVac®-based vaccines are expected to
deliver consistent results in clinical studies.
The adenovirus production process, including down-stream processing,
results in purified mature recombinant adenovirus. This product expectedly
contains a certain level of impurities, either process-related or product-related.
Product-related impurities are defined as the percentage of virus subpopulations
such as aggregates and immature or defective particles against intact virus. The
presence of such impurities could initiate host immune response leading to
adverse reactions. Moreover, the presence of immature particles can either
enhance or inhibit transduction efficiencies. To ensure the safety, efficacy, and
quality of the vaccine, advanced measurement methods are needed to monitor the
relative quantities of intact virus and product-related impurities [4].
Purity of an adenovirus can be determined by cesium chloride (CsCl) density
gradient centrifugation. In this method, the complete virus is separated from
incomplete particles based on their density [5]. This method is only a qualitative
preparative assay [6, 7]. Differential centrifugal sedimentation (DCS) is another
method to characterize particle size distribution by utilizing sucrose gradient
Purity determination of adenoviruses
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solutions [8]. The adenoviruses will sediment differently in a sucrose gradient
according to their size shape and density. In addition, an analytical ultra
centrifugation (AUC) method to determine the purity of adenovirus has been
reported by Berkowitz and Philo [9, 10]. AUC separation is based on differences in
sedimentation velocity and has the advantage of being a quantitative assay. The
disadvantages of this method are its cost, low throughput and an experienced
operator is required to operate the AUC. Hence, the use of AUC as a routine assay
is limited.
Recent studies have shown that column chromatography is a most versatile
and powerful method for adenovirus purification and quantification [11, 12].
Column chromatography may utilize different columns to achieve the desired level
of purification. For example, ion exchange [11, 13, 14], size exclusion
chromatography [12], and affinity chromatography [15], are becoming useful tools
for virus purification [12].
Capillary electrophoresis (CE) is another method that can be used to
analyze viruses. Several groups have investigated human rhinoviruses (HRVs) using
capillary zone electrophoresis (CZE) [16-18]. Mann et al. has developed a method
to separate recombinant adenovirus type 5 in 25 mM sodium phosphate buffer pH
7.0 using a capillary coated with polyvinyl alcohol (PVA) [19].
The aim of this study was to find suitable analytical methods for assessing
the purity of adenovirus preparations. A suitable method is rapid, reliable, and
informative about the quantity and quality of the different populations of
adenovirus particles. In this research, we explored chromatographic methods, such
as size-exclusion chromatography (SEC), ion exchange chromatography (IEX) and
reversed-phase chromatography (RP-HPLC) to determine the purity of adenovirus.
Capillary electrophoresis was also used to in this study, as its short analysis time
and high-efficiency for separations of macromolecules with minimum sample
preparation and sample consumption may be an important advantage [20, 21].
Differential centrifugal sedimentation (DCS) and analytical ultracentrifugation
(AUC) were used as orthogonal techniques to the chromatographic methods.
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THEORY
2.1 Adenovirus
Adenovirus is a non-enveloped, isocahedral DNA virus that belongs to
adenoviridae family [2, 3]. It is composed of an outer capsid surrounding an inner
DNA core with a particle size of approximately 80 nm and a molecular mass of
about 150 MDa [22, 23]. The mass is determined by the protein content (87%) and
DNA content (13%) [9]. Adenovirus proteins can be subdivided in three major
groups: major capsid proteins, minor proteins, and the core proteins. Figure 1
provides a schematic diagram of an adenovirus [2].
Figure 1: Schematic overview of adenovirus. Adenovirus proteins are divided in three major groups: major capsid proteins [hexon (II), penton base (III), fibre (IV)], minor proteins, and core proteins. The virus DNA, indicated by black thick line, is associated with core proteins. (Ref. [2])
The major capsid proteins are the hexon, penton-base, and fiber [3]. There
are 240 homotrimetric hexons (pentamers of protein II) and 12 pentons that
consist of 12 penton bases (protein III), and 12 extended fibers (trimers of protein
IV) [2, 3, 23]. Penton bases and fibers are involved in mediating adenovirus
infection. Protein IIIa, VI, VIII, and IX are minor components which are also
Purity determination of adenoviruses
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associated with the capsid and located near the premises of major capsid proteins.
Protein VI is involved in facilitating endosomal escape by disruption of the
endosomal membrane and the main function of protein VI is to facilitate nuclear
import of hexon proteins [23]. Mutant virus analysis suggests that protein VIII plays
a role in the capsid structural stability. The precursor of protein VIII is undetectable
in complete particles while it is present in empty capsids or incomplete particles.
Protein IX acts as capsid cement. In addition, protein IX also affects the DNA-
packaging capacity of human adenovirus and transcriptional activity of several
promoters [23]. Six proteins are situated in the virus core. Five of the components
(V, VII, Mu, IVa2, and terminal protein) are associated with the double stranded
DNA genome, while the 23KDa virus protease plays an important role in the
assembly of the virus [2].
A purified adenovirus preparation consists of monomer adenovirus (particle
size 77 nm) and aggregates [24]. The aggregates are reversible and high ionic
strength conditions can induce dissociation of the aggregates [4]. It was shown in
pH stability data of the adenovirus that adenovirus type 5 spontaneously
dissociated at pH lower than 5.8 [25]. A small increase in the electrophoretic
mobility of adenovirus type 5 was observed by increasing the pH between 5.8 and
8.3 and the mobility stabilizes between pH 8.3 and 9.0 [19]. Based on pH stability
data and the electrophoretic mobility experiment, the pI value of adenovirus type
5 is predicted around 5.8 [19]. Other serotypes of adenovirus may have different pI
value dependent on their peptide compositions [26].
Empty capsids of adenoviruses are detected after the purification of
recombinant adenovirus. These empty capsids differ from complete virus in density
and DNA content. Due to the absence or lack of DNA, empty capsids have lower
density (approximately 1.29-1.30 g/mL) than intact adenovirus (1.34 g/mL) [5, 6].
Empty capsids generally contain hexon, penton and fiber, while the core proteins V
and VII are missing. Other proteins that are not present on the complete virus may
be observed in empty capsids. Some of the proteins have been identified as the
Purity determination of adenoviruses
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precursor proteins, pVI and pVIII, or scaffolding proteins L1 52/55K [5]. Protein
52/55K is required for the infectious virus assembly [27].
2.2 Method for Analysis of Adenovirus Purity and Product-Related Impurities
2.2.1 UV absorbance
The complete virus, empty capsids, and aggregation can be analyzed by UV
absorbance at 260, 280, and 320 nm. The ratio A260/A280 shows the relationship
between nucleic acid and protein. A pure adenovirus type 35 shows an A260/A280
ratio of 1.2-1.3. Lower ratios of A260/A280 are typical for protein, while higher ratios
of A260/A280 indicate that the adenovirus preparation could be contaminated with
non viral DNA [28]. The A320/A260 ratio reflects the presence of aggregates in the
purified virus preparation. A typical range of A320/A260 for purified virus is 0.22-0.27
and can rise rapidly to 0.3-0.7 as virus aggregation is initiated [5].
2.2.2 Ultracentrifugation
Ultracentrifugation is a purification method to separate macromolecules,
such as DNA and viruses, based on their partial specific volume (combination of
density, size and shape). Macromolecules are separated from contaminants by
ultracentrifugation in a density gradient. For viruses, salts such as Cs2O4 or CsCl are
used to form the density gradient [7].
For adenoviruses, a sample is applied to a step gradient of CsCl where the
density of the bottom and the upper layer of CsCl are 1.4 g/mL and 1.25 g/mL,
respectively. Subsequently, the density gradients containing the sample are
centrifuged at a certain speed for a period of time. As a consequence, the virus
separates from cellular debris and collects in a band between the two CsCl layers.
This collected band is then mixed with CsCl at 1.35 g/mL and centrifuged overnight.
As a result, the intact virus is separated from incomplete particles (Figure 2). The
CsCl layers are collected from the tubes and CsCl can be removed by dialysis. Intact
viruses will be in the lower band since they have a higher density compared to
Purity determination of adenoviruses
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incomplete and empty particles, whereas incomplete particles are present in the
upper band. This method is easy to perform and yields high-purity virus
preparations [6].
Figure 2. Example of recombinant adenovirus particles density forms separated by preparative CsCl gradient centrifugation. Upper light blue band contains incomplete particles; lower thick band contains intact virus. (from Ref. [29])
2.2.3 Differential Centrifugal Sedimentation (DCS)
Differential Centrifugal Sedimentation (DCS) is a method to determine the
particle size and mass distribution based on sedimentation of particles. Stokes Law
can be used to determine the spherical particle size from measurement of the time
needed for particles to settle a known distance in a liquid of known density and
viscosity. Sedimentation can either be based on gravitational or centrifugal force
[8]. Gravitational force is used to measure large particles. For small particles, like
virus, centrifugal sedimentation is preferably used to determine the size
distribution (the high centrifugal force makes the sedimentation of small particles
faster than Brownian motion so the particles start to settle according to Stokes
Law).
For this study, differential sedimentation was used. Samples are placed on
the top of a clear fluid and subjected to a centrifugal acceleration to yield particle
sedimentation [30]. Initially, the detector reads at maximum intensity that
decreases when particles pass the detector beam. The signal reduction is
proportional to the particle concentration when the X-ray detector is used. For
Purity determination of adenoviruses
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monochromatic light source, Mie theory light scattering is used to estimate the
particle concentration [31].
DCS instruments use a hollow, optically clear disc that is driven by a motor
(Figure 3). A short wavelength (400-500 nm) monochromatic light beam is used as
detector beam, as it gives good sensitivity for measuring particles smaller than 100
nm diameter [8]. The DCS operation is very simple, and multiple measurements
can be done in one day. Furthermore, this method can produce accurate and
reproducible result in a short time. However, DCS cannot be used for quantitative
assay of the incompletes particles because of the difficulty to know the exact
values of refractive index, shape and particle density of incomplete particles.
Moreover, the DCS software cannot be qualified for Good Manufacturing Practices
(GMP) purposes so this is difficult to implement in quality control laboratories [32].
Figure 3. A typical DCS design. (Left) Front view; (Right) Side view (Ref. [8])
2.2.4 High Performance Liquid Chromatography (HPLC)
HPLC is a separation method based on the partition of sample compounds
between a solid stationary and a liquid mobile phase under high pressure [33]. It
potentially gives rapid separation of different sample compounds with an
extraordinary peak resolution. The highest efficiencies can be achieved by the use
of very small particles of stationary phase and high pressure to maintain a high
flow rate [12, 33].
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Many HPLC methods are developed for analyzing adenoviruses, such as ion-
exchange chromatography [11, 13, 28, 34, 35], size exclusion chromatography [12],
and reversed-phase chromatography [36, 37]. The modes of chromatography that
were explored in this study for assessing the purity of viral particles are size
exclusion chromatography (SEC), reversed phase high-performance liquid
chromatography (RP-HPLC), and ion exchange chromatography (IEC).
2.2.4.1 Size Exclusion Chromatography (SEC-HPLC)
Size-exclusion chromatography (SEC), also known as gel-filtration
chromatography, is a technique for separating analytes based on their size or
hydrodynamic volume. This technique can be used to analyze biological samples
that consist of proteins with different molecular weights [12, 38]. The solid-phase
matrix consists of porous beads packed into a glass or steel column so the mobile
phase can diffuse to the volume inside the pores and outside the beads. High
porosity beads lead to a total liquid volume of > 95% for packed columns [38].
In SEC (Figure 4), the pore structure of the resin provides a molecular sieve;
smaller molecules penetrate into the pores of the beads and larger molecules
remain outside of the beads as they are unable to enter the pores [7, 38, 39]. The
order of elution can be predicted by the size of the molecules, i.e. large molecules
elute first from an SEC column (as they are excluded from the pores) followed by
small molecules [12, 38, 39].
Figure 4. Schematic overview of a size exclusion column. Larger particles will elute first followed by smaller particles (Ref. [40])
Purity determination of adenoviruses
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There are various types of stationary phase for SEC. The choice of stationary
phase depends on the molecular weight and the solubility of the analytes in the
sample. Silica porous beads are suitable for protein separations whereas polymer
porous beads are designed for organic polymers, poly- and oligosaccharides, and
viruses. Zhang et al., have developed a method for assessing adenovirus type 5
purity using Bio-Sep-Sec-S3000 column. The method allowed to quantify the
adenovirus and to detect impurities [12].
2.2.4.2 Ion Exchange Chromatography (IEX-HPLC)
Ion exchange chromatography is a separation process based on differences
in net surface charge of the molecules. This technique is appropriate for analysis of
proteins, oligonucleotides, nucleic acids or other charged biomolecules. There are
four steps during the separation process in IEX. The first step is the equilibration of
the column to the appropriate pH and salt concentration. The second step is the
sample loading, by which the analyte of interest will bind to the column. The third
step is elution of the analytes from the column by changing the pH or increasing
the salt concentration. The last step is column regeneration. IEX can be classified
into anion exchange and cation exchange (Figure 5). In cation exchange, a
negatively charged group in the stationary phase is used to attract positively
charged molecules. Sulfonic acid, phenolic hydroxyl and carbonyl are commonly
used as cation exchangers. The former is known as strongly acidic cation exchanger
while the others are weakly acidic cation exchangers. In anion exchange, negatively
charged molecules are attracted into positively charged groups in the stationary
phase. Quartenary, aromatic and aliphatic amino groups are examples of anion
exchangers [41, 42]. Separation is achieved as differently charged molecules have
different degrees of interaction with the ion exchanger. The separation can be
optimized by varying the pH and/or ionic strength of the mobile phase, or by
eluting in a pH or salt gradient [42, 43].
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Figure 5. Schematic of Ion-exchange chromatography. (Left) Anion Exchange column; (Right) Cation Exchange column. (from Ref. [44])
Several publications have been published on the use of ion-exchange
chromatography for analysis of adenoviruses. Uno-Q column [28, 34] and Q
Sepharose XL column [13, 35] have been used for quantifying adenovirus type 5.
Bio-Monolith QA column can be use to separate intact adenovirus type 5 from
contaminants [11].
2.2.4.3 Reversed Phase HPLC (RP-HPLC)
In reversed phase mode, a non-polar compound is utilized as the stationary
phase and a polar solvent is used as mobile phase. The separation mechanism is
based on the partitioning (differential affinity) of the analytes between stationary
and mobile phases.
Stationary phases of reversed phase columns are traditionally made up of
silica modified with alkyl chains of a certain length. The most common alkyl chains
are C2, C4, C8, C18. Phenyl can also be used as aromatic side chain. Short alkyl chains
are suitable for the separation of more polar analytes whereas longer alkyl chains
are preferred for moderately polar and non-polar analytes separation [42, 45].
Elution in RP-HPLC can be isocratic or in a gradient condition. In isocratic
elution, the concentration of the mobile phase remains constant during the
analysis. In gradient elution, the concentration of the organic solvent is increased
over a period of time. The analytes elute in order of increasing molecular
hydrophobicity [42].
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Lehmberg et al., have successfully quantified adenovirus type 5 by RP-HPLC
through quantification of the structural proteins [36]. Takahashi and co-workers
were also able to quantify the empty capsids of adenovirus type 5 by RP-HPLC.
They used precursor protein VII as the marker for quantifying the empty capsids
[37].
2.2.5 Capillary Electrophoresis (CE)
Capillary electrophoresis (CE) is a very efficient separation technique
available for the analysis of both small and large molecules [46]. Capillary
electrophoresis is defined as the differential movement of charged particles under
the influence of an electric field [47, 48]. A typical CE system, which is illustrated in
Figure 6, consists of a high-voltage power supply, a sample introduction system,
two buffer reservoirs, two electrodes, a capillary, a detector and an output device.
Each side of the high power supply is connected to an electrode. These electrodes
help to induce an electric field to initiate the migration of the sample from the
anode to the cathode through the capillary [49]. The capillary is filled with the
desired buffer solution and the ends of capillary are dipped in vials containing the
same buffer solution [50]. As the separation in CE depends not only on the
electrophoretic mobility but also on the viscosity of the solution, a temperature
control device is needed to ensure reproducible results. Most CE instruments make
use of forced air and liquid to control the temperature.
Figure 6. The instrumental set-up of CE system
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Capillary electrophoresis can be operated in several modes. Examples of CE
modes are capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE),
micellar electrokinetic capillary chromatography (MEKC), capillary
electrochromatography (CEC), capillary isotacophoresis (CITP), and capillary
isoelectric focusing (CIEF). In this research, only CZE was used.
Capillary zone electrophoresis (CZE) or free solution capillary
electrophoresis is the most common form of CE. The separation of a mixture in a
solution is based on the differences in electrophoretic mobility and the
electroosmotic flow (EOF). CZE is suitable for separation of both anionic and
cationic analytes. Neutral molecules are not separated based on electrophoretic
mobility and will migrate together with the EOF [48].
Electrophoresis
Electrophoresis is the migration of sample ions in a solution under the
influence of an electric field. The rate of mobility is directly proportional to the
applied electric field; the greater the strength of electrical field, the faster the
mobility. Furthermore, the electrophoretic mobility depends on the charge of the
molecule, the solution viscosity and the molecule’s radius. If two ions have the
same size, the one with greater charge will have a greater mobility. For ions with
the same charge, the smaller particle has less friction and will migrate at a faster
rate [51].
Electroosmotic Flow (EOF)
A fundamental characteristic of CE operation is the electroosmotic flow
(EOF). EOF is the bulk flow of solution caused by the application of a voltage to an
electrolyte-filled capillary (Figure 7). The flow occurs when the buffer (pH greater
than 3) running through the capillary so the free silanol groups are deprotonated
[47, 49]. As the whole system must be electrically neutral, the buffer solution will
form a diffuse double-layer close to the wall. The inner layer is fixed, while the
Purity determination of adenoviruses
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outer layer is mobile. When a voltage is applied to the capillary, the mobile layer
will move toward the cathode and creating an EOF [47, 49].
Figure 7. The principle of electroosmosis (from Ref. [52])
The advantage of the EOF is the flat flow profile in the capillary. There is no
pressure drop within the capillary because the electric force of the flow is
uniformly distributed along the capillary. As a result, no peak broadening is caused
by the EOF [47, 49].
Purity determination of adenoviruses
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EXPERIMENTAL SECTION
3.1 Standard and Samples
A specific adenovirus type was used as control sample. CsCl fractions (upper
and lower band), obtained by ultracentrifugation, were used as samples. The upper
band represents incomplete particles (impurities of adenovirus), while the lower
band consists of intact particles of adenovirus. All samples were thawed slowly at
room temperature prior to analysis.
3.2 Differential Centrifugal Sedimentation
DCS analysis of adenovirus was performed using a CPS instrument model
DC24000 equipped with a detector. 200 µL of each adenovirus control sample and
samples was injected into the DCS instrument. Samples were injected as received
(no sample treatment was required for this analysis).
3.3 Size Exclusion Chromatography
3.3.1 Materials and Methods
An Alliance 2695 HPLC system with a PDA detector was used for the SEC
experiment. Silica- and polymer-based columns were utilized as analytical columns.
To determine the optimal conditions for purity determination of adenovirus,
buffers with different pH values were used as mobile phase. The injection volume
was 50 µL.
3.3.2 Preparation of Adenovirus Control Sample and CsCl Fractions
A specific adenovirus was used as control sample and CsCl fractions (upper
and lower band) were used as samples. All samples were thawed slowly at room
temperature. Control sample and samples were transferred into HPLC vials and
placed in the autosampler for analysis.
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3.4 Anion Exchange Chromatography
3.4.1 Materials and Methods
Anion-exchange chromatography was performed on a quaternary column.
An Alliance 2695 HPLC system with a PDA detector was used. Chromatograms were
extracted from the PDA data. Buffers with gradient elution were used as mobile
phase. The injection volume was 100 µL.
3.4.2 Preparation of Adenovirus Control Sample and CsCl Fractions
Adenovirus control sample and CsCl fractions are prepared as in 3.3.2.
3.5 Capillary Electrophoresis
3.5.1 Materials and Methods
CE analysis was carried out using an Agilent CE (G7100A). The different
capillaries were used for the experiments. Buffers with different pH values were
used as BGE.
3.5.2 Preparation of Adenovirus Control Sample and CsCl Fractions
A specific adenovirus control sample and CsCl fractions were thawed slowly
at room temperature.
3.6 Adenovirus Purity Determination Using Reversed-Phase HPLC
3.6.1 Materials and Methods
The RP-HPLC experiments were performed on an Alliance 2695 HPLC
system equipped with a PDA detector. C4 column was used for separation. The
mobile phases were eluted with the gradient program.
Purity determination of adenoviruses
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3.6.2 Preparation of Adenovirus Control samples and CsCl Fractions
The adenovirus control sample and the CsCl fractions were thawed slowly
at room temperature. The upper and lower CsCl fractions were mixed to have
certain amount of the incomplete particles.
Purity determination of adenoviruses
24
RESULTS AND DISCUSSION
4.1 Differential Centrifugal Sedimentation (DCS) and AUC
The adenovirus control sample and the CsCl fractions were analyzed by DCS
using CPS instrument. The result is shown in Figure 8. The adenovirus control
sample showed 2 peaks. The small peak represents incomplete particles, with an
estimated diameter approximately 65 nm, whereas the higher peak represents
complete particles with an estimated diameter of about 69 nm. The upper
(incomplete particles) and lower (complete particles) band of CsCl fractions
showed differences in particle diameter. The peaks of the upper band and lower
band were detected at approximately 65 nm and approximately 71 nm,
respectively, overlapping with the two peaks of the adenovirus control sample.
Figure 8. Result of DCS analysis (–) Adenovirus control sample; (−) lower band of CsCl fraction; (–) upper band of CsCl fraction.
DCS result was confirmed by AUC (Figure 9). The AUC results further
showed that there were complete particles in the upper band (see peaks between
600 and 700 s in the red trace in Figure 9).
Purity determination of adenoviruses
25
Figure 9. AUC results: Sedimentation distribution of (–) adenovirus control sample, (–) upper band of CsCl fraction and (–) lower band of CsCl fraction.
The DCS and AUC results indicated that the incomplete/empty and
complete particles can be distinguished based on their density. This was an
expected result as these different types of particles can also be separated by CsCl
density gradient ultracentrifugation. The disadvantages of the DCS technique as a
method for analysis of product-related impurities are that it is not quantitative and
the instrument cannot be operated in a GMP laboratory. This is also a drawback of
the AUC technique. Although AUC does yield quantitative result, it requires an
experienced operator. Furthermore, the sample throughput of AUC is low
compared to HPLC. Therefore, the feasibility of HPLC methods for the purpose of
this analysis was explored.
4.2 Size Exclusion Chromatography (SEC-HPLC) Result
An alternative method to determine quantitavely the purity of adenovirus is
needed since AUC is not the method of choice, as explained above. Size exclusion
chromatography (SEC) was chosen as the first chromatography method for
determining the purity of adenovirus since adenoviruses are already purified using
Purity determination of adenoviruses
26
membrane-based anion-exchange in Downstream Process (DSP). A different
chromatographic mode of separation, such as size exclusion, is expected to resolve
the impurities in adenovirus products better than an analytical method with the
same mode of separation as that used for purification. The adenovirus control
sample was initially analyzed on a column, which has a pore size of 200 nm and is
made from silica. The chromatograms of the adenovirus control sample in Figure
10 show that buffer pH 1 gives a good peak response of adenovirus. Fixing this pH,
the flow rate and column temperature were optimized. The result is shown in
Figure 11.
Figure 10. Chromatograms of adenovirus control samples at different pH values. buffer (–) pH 1;
(–) pH 2; (–) pH 3.
Purity determination of adenoviruses
27
Figure 11. Chromatograms of adenovirus control samples in pH 1 (–) default condition; (–)
condition 3; (–) condition 2; (–) condition 3; (–) blank.
Figure 11 shows that default condition is the best separation conditions for
adenovirus. However, a blank peak eluted at the same retention time as the
adenovirus control sample, as observed in the chromatogram. The result was
unexpected since blank has particle size smaller than the adenovirus. The
unexpected result could be an indication that the column is damaged.
Furthermore, the repeatability of adenovirus peak height between injections was
not good (data not shown). We hypothesized that the poor repeatability was
caused by strong interaction between adenovirus and silica. This assumption was
reinforced by the publication of Mann et al., which indicated that adenovirus are
adsorbed at silica surfaces [19]. Since the silica column interacts with adenovirus
and resulted in poor reproducibility, a different SEC column was needed.
Polymer column with a pore size 100 nm was chosen for the next SEC
experiment. This column was used successfully for analysis of other viruses in-
house and it was expected that the column would also work for adenovirus. Based
on the method used in-house for analysis of other viruses, buffer pH 8.0 containing
salt was used as mobile phase. The purpose of adding salt is to minimize
hydrophobic interactions between the polymer matrixes and adenovirus which
could lead to low recovery and carry-over effects. The adenovirus eluted at 13 min
Purity determination of adenoviruses
28
and was well separated from the blank peak (Figure 12). Six injections of
adenovirus control sample showed a good repeatability of peak area (RSD < 5%).
No carry over was observed in the blank (black trace in Figure 12).
Figure 12. SEC chromatogram of adenovirus control sample in polymer-based column. Adenovirus
eluted at 13 min while the blank peak eluted at 20 min. (–) Adenovirus; (–) Blank.
Unfortunately, injections of the upper and lower band of the CsCl gradient
showed similar retention times (Figure 13). These results were unexpected
because we had determined by DCS and AUC that the lower (complete particles)
and upper (incomplete particles) bands of CsCl fractions consisted of particles with
different particle sizes (based on their separation by density). It is possible that the
polymer-based column is not able to resolve the small particle size differences
between incomplete particles and complete particles of adenovirus.
Purity determination of adenoviruses
29
Figure 13. SEC chromatograms of adenovirus in polymer-based column. (−) adenovirus control sample; (−) the lower band of CsCl fractions; (−) the upper band of CsCl fractions; (−) blank.
Even though the particles of the two CsCl fractions cannot be separated
using SEC-HPLC method, this technique can still be used for quantification of
adenovirus particles in purified samples.
4.3 Anion-Exchange Chromatography (AEX-HPLC) Result
Anion-exchange analytical column was explored to determine the purity of
adenovirus. 100µL samples were injected and eluted in an increasing salt gradient
with mobile phase B. Figure 14 shows the AEX chromatograms.
Purity determination of adenoviruses
30
Figure 14. AEX-HPLC chromatograms of (−) adenovirus control sample; (−) upper band and (−) lower bands of CsCl gradient. The upper and lower bands of the CsCl gradient have similar retention times, while the retention time of control sample is slightly different, which is explained by the differences in inserted gene for these two types of samples.
The chromatograms clearly show that there is no significant difference in
retention time between the upper and lower bands of the CsCl gradient. The
complete and incomplete/empty particles eluted at 11.27 min and 11.30 min,
respectively. The adenovirus control sample eluted at 11.52 min. The retention
time difference between the control sample and the CsCl gradient fractions can be
explained by the different inserted genes in these adenovirus samples. The results
for the upper and lower bands indicated that the incomplete/empty and complete
adenovirus particles are similarly charged at the pH of the mobile phase used, so
they cannot be distinguished from each other by AEX-HPLC. An investigation on the
effect of mobile phase pH on the resolution between the upper and lower bands
may be useful for further development.
4.4 Capillary Electrophoresis (CE) Result
CE was chosen as an alternative method since SEC cannot resolve
incomplete/empty from complete adenovirus particles, and only a slight difference
in retention times was observed by AEX-HPLC. CE is expected to separate particles
based on their charge to mass ratios with resolution higher than AEX-HPLC.
Purity determination of adenoviruses
31
In the first run, the experimental condition were according to Mann et al.
[19] with slight modifications. In addition, different capillaries, pH values and
molarities were evaluated to determine the optimum condition for adenovirus
separation. Prior to the experiments, a range of different voltages was applied to
the background electrolyte (BGE) to observe excessive Joule heating effect by
plotting the applied voltage and the measured current (Table 1).
Table 1. A range of different voltages was applied to the BGE for observation of Joule heating effect
BGE
Voltage
-5 -10 -15 -20 -25 -30
Current (µA)
BGE 1 9 19 30 43 59 79
BGE 2 30 65 NA NA NA NA
BGE 3 7 13 20 28 38 49
BGE 4 11 22 35 51 68 NA
BGE 5 7 13 20 28 37 48
Applied voltages that still showed linear correlation with the current were
chosen as applicable voltages in order to have good efficiency and resolution. The
maximum applicable voltage for each BGE is presented in Table 2. Separation was
done in reversed polarity as adenovirus is negatively charged at pH above 6.0. BGE
2 was not used for the experiment due to low applicable voltage that would result
in longer analysis time for adenovirus.
Table 2. Maximum applied voltage for BGE solutions
No Background Electrolyte (BGE)
solution
Maximum applied voltage
( - kV)
1 BGE 1 20
2 BGE 3 25
3 BGE 4 18
4 BGE 5 25
Purity determination of adenoviruses
32
4.4.1 Capillary 1
The electropherograms of adenovirus and CsCl fractions in each BGE
obtained with capillary 1 are shown in Figure 15 to Figure 18. For all samples, the
adenovirus peak eluted in less than 5 min in all BGE. No differences in migration
times were observed between the CsCl fractions and between these fractions and
the adenovirus control sample.
Figure 15. Electropherograms of different adenovirus samples. Electrophoresis in BGE 1; 20 kV in reverse polarity. (−) upper band of CsCl gradient; (−) lower band of CsCl gradient (−) adenovirus control sample.
Figure 16. Electropherograms of different adenovirus samples. Electrophoresis in BGE 3; 18 kV in reverse polarity. (−) upper band of CsCl gradient; (−) lower band of CsCl gradient. (−) adenovirus control sample.
Purity determination of adenoviruses
33
Figure 17. Electropherograms of different adenovirus samples. Electrophoresis in BGE 4; 25 kV in reverse polarity. (−) upper band of CsCl gradient; (−) lower band of CsCl gradient; (−) adenovirus control sample.
Figure 18. Electropherograms of different adenovirus samples. Electrophoresis in BGE 5; 25 kV in reverse polarity. (−) upper band of CsCl gradient; (−) lower band of CsCl gradient; (−) adenovirus control sample.
Purity determination of adenoviruses
34
4.4.2 Capillary 2
The separation of complete and incomplete adenovirus particles in capillary
1 was unsuccessful. Therefore, another experiment was done using capillary 2. This
capillary was successfully used by Mann et al. to analyze recombinant adenovirus
type 5. The separation of adenovirus in capillary 2 was evaluated in BGE as used for
capillary 1. The maximum applicable voltages were the same as for capillary 1 and
no significant differences in migration times were observed for each BGE value.
The results of adenovirus analysis in capillaries 1 and 2 are summarized in Table 3.
The unknown peaks, which looked like electrodispersion phenomenon,
were observed in the adenovirus control sample and the CsCl fractions when BGE 3
and 4 was used as BGE in the capillaries 1 and 2. We attributed those peaks to pH
instability.
Table 3. Migration times of adenovirus in capillaries 1 and 2
BGE Applied
Voltage
(- kV)
Migration Times
(Minutes)
Resolution
complete vs
incomplete
Cap. 1 Cap. 2 Cap. 1 Cap. 2
BGE 1 20 3.2 3.3 No No
BGE 2 5 - - No No
BGE 3 25 2.1 2.2 No No
BGE 4 18 3.4 3.6 No No
BGE 5 25 2.5 2.2 No No
In conclusion, capillaries 1 and 2 were not able to resolve the CsCl fractions.
The adenovirus control sample and the CsCl fractions showed similar migration
times in both capillaries 1 and 2.
4.4.3 Capillary 3 with dynamic coating
A capillary 3 dynamically coated with buffer pH 6.0 and 7.1 was tested. The
different coating mode was expected to be able to separate complete and
incomplete particles. The electropherograms of adenovirus at pH 6.0 and pH 7.1
are shown in Figure 19 and Figure 20, respectively.
Purity determination of adenoviruses
35
Figure 19. Electropherograms of different adenovirus samples with dynamic coating. BGE: buffer pH 6.0. (−) adenovirus control sample; (−) lower band of CsCl gradient; (−) upper band of CsCl gradient.
Figure 20. Electropherograms of different adenovirus samples with dynamic coating. BGE: buffer pH 7.1. (−) adenovirus control sample; (−) lower band of CsCl gradient; (−) upper band of CsCl gradient.
The peaks of adenovirus control sample and CsCl gradient fractions were
split at pH 6.0, and only the adenovirus control sample showed a peak at pH 7.1.
The CE results using capillary 3 were inconclusive due to a broken electrode.
None of CE experiments led to satisfactory results because the
incomplete/empty particles could not be distinguished from the complete particles
Purity determination of adenoviruses
36
under the tested conditions. It seems that the charge to mass ratio of adenovirus in
these different types of particles is only slightly different. As a result, a different
technique is needed to determine the concentration of incomplete particles. The
CE method, in which a capillary 1 with BGE 1 and BGE 5 was used, may still be used
for quantification of adenovirus particles.
4.5 Reversed-Phase High Performance Liquid Chromatography (RP-HPLC)
An RP-HPLC method has been developed for stability and purity testing of
adenovirus control sample but this method has only been used for qualitative
purposes. The CsCl fractions containing incomplete/empty particles and complete
particles have not yet been analyzed by this method. The conditions of RP-HPLC
disrupt the adenovirus particles and allow for the separation of the individual
adenovirus proteins. Representative chromatograms of the upper and lower bands
of CsCl gradient are presented in Figure 21.
Figure 21. RP-HPLC chromatogram overlay of: (−) upper band and (−) lower band of CsCl gradient. The main differences between the chromatograms are marked with arrows.
As it can be seen in Figure 21, differences were observed between the
chromatograms of the upper and lower bands. The peak areas of some proteins
(protein 1, 5, 7 and 8) were decreased in the upper band whereas other proteins
(protein 3, 9, 12, and 13) were increased in the upper band, compared to the lower
band of the CsCl gradient. Proteins 2, 6 and 10 had the same peak areas in the
Purity determination of adenoviruses
37
upper and lower bands. In addition, two unknown peaks were observed in the
upper band of the CsCl gradient. The decreased peak area of the DNA-associated
proteins in the upper band is understood, as this band consists of
empty/incomplete particles. The absence of protein 9 in the lower band is
expected as this protein is not supposed to be present in complete adenovirus
particles. Interestingly, the protein 13 was also mainly present in the fraction
corresponding to empty/incomplete particles. Protein 13 is related with the
infectious virus assembly and it is also required for the encapsidation of the
genome. Predominance of the protein 13 in the upper band indicated that this
fraction contained virus particles which are still being formed or for which genome
encapsidation is ongoing, in agreement with the expectation that this fraction
contains mainly empty/incomplete adenovirus particles [53].
In order to determine if there is a linear relationship between the relative
peak area of the protein 13 and the percentage of empty/incomplete particles,
different ratios of the upper and lower band were mixed as described in section
3.6.2. The final virus particle concentrations of the mixed samples are to be
considered as estimations. The concentration of empty/incomplete particles can
be calculated according to Vellekamp et al. [5], but this calculation still needs to be
validated before it can be considered reliable.
The AUC data were used to estimate the percentage of complete particles
that were present in the upper band of CsCl gradient. Based on the AUC relative
peak areas, there were 17 % of complete particles in the upper band. The
percentages of empty/incomplete particles in the mixed samples were corrected
by this factor. It is stressed that these percentages remain estimations, as the
quantitative ability of the AUC method has not yet been demonstrated. Linear
correlations for each protein were observed by plotting the percentage of
incomplete/empty particles versus peak area. The RP-HPLC chromatograms are
shown in Figure 22.
Purity determination of adenoviruses
38
Figure 22. RP-HPLC chromatograms of samples with varying concentrations of empty/incomplete particles obtained by mixing bands of the CsCl gradient.
For this report, protein 13 was chosen as marker of empty/incomplete
particles because it is only expected to be present in particles that are being
assembled or filled with viral DNA. Table 4 shows that the peaks assigned to
proteins 2, 6 and 10 were the same for all the mixed fractions. The % RSD value of
protein 10 peak area for duplicate analysis of the same sample is comparable to
the % RSD calculated for all the mixed samples, indicating that the relative
concentration of this protein is the same for all types of virus particles (empty,
incomplete and complete). It is expected for protein 10 to be present in equal
concentrations in all types of adenovirus. Therefore, protein 10 was chosen as
internal control peak. The areas of the other peaks in the chromatogram can then
be reported relative to peak 10 in order to eliminate possible result artifact due to
variations in sample concentration (VP/mL).
Purity determination of adenoviruses
39
Table 4. The peak areas and RSD value of protein 2, 6 and 10 in all mixed fractions of incomplete particles.
Protein n=2 % of empty/ incomplete particles RSD
(%) 0% 21% 42% 63% 83%
2 Peak Area 20829 20569 19327 19565 17280 7.19
RSD (%) 1.4 0.3 1.3 0.1 2.1
6 Peak Area 27820 27900 27160 27110 26005 2.79
RSD (%) 0.7 0.9 0.9 1.0 0.0
10 Peak Area 25710 26160 25560 26372 25646 1.37
RSD (%) 0.9 0.8 1.5 1.3 1.1
Quantitation of Proteins Using HPLC-Detector Response
The molar concentration of proteins were calculated based on papers from
Eberlein and Lehmberg [36, 54]. According to Eberlein [54], the detector response
factors from HPLC could be used to measure the protein concentration of
adenovirus. The peak area, peak height, flow rate, extinction coefficient and
molecular weight data were needed for the calculations of protein concentration.
The extinction coefficients and molecular weights of proteins 10 and 13 were
obtained from ProtParam tool, ExPASy [55].
The detector responses were calculated in two steps as follows:
A. Peak height response factor from extinction coefficient (Rfh)
The response factor for the peak height (Rfh) was calculated as in Equation 1.
Rfh = absorbance (at 1 AU) x extinction coefficient Equation 1
The theoretical extinction coefficients for proteins 10 and 13 were 1.142 and
0.846, respectively. The absorbances of proteins 10 and 13 at 1 AU and the
responses factor for the peak height (Rfh) in the different mixed samples are listed
in Table 5 and Table 6.
Purity determination of adenoviruses
40
Table 5. Absorbances of proteins 10 and 13
% of empty/incomplete particles Absorbance at 1 AU
Protein 10 Protein 13
0% 892 926.3
21% 908 984.3
42% 924 1006
62% 938.5 985
83% 934 1018.2
Table 6. Response factors for peak height (Rfh) of proteins 10 and 13
% of empty/incomplete particles Rfh (mV)
Protein 10 Protein 13
0% 1018.6 783.6
21% 1036.9 832.7
42% 1055.2 851.1
62% 1071.8 833.3
83% 1066.6 861.4
B. Peak area response factor (RfA) from extinction coefficient
The peak area response factor (RfA) could be obtained from the extinction
coefficient by integrating the peak height (Rfh) over its duration (i.e calculating the
peak area) [Equation 2]. The duration is determined by the flow rate ‘f’ (ml/60s).
RfA = Rfh x f -1 Equation 2
From the value of RfA, the eluted quantity (q) is calculated as in Equation 3:
q = Peak area x RfA-1 x d-1 Equation 3
where q = eluted quantity (mg), RfA = area response factor for peak area at f =0.2
ml/min, d = detector cell path length (1 cm). The molar concentration is calculated
by dividing the eluted quantity (mg/mL) by the molecular weight of each protein.
The measured peak area response factors (RfA), the injected quantities and
the molar concentrations are listed in Table 7.
Purity determination of adenoviruses
41
Table 7. Peak area response factors, injected quantities and molar concentrations of protein 10 and 13 in the different mixed samples of upper and lower bands from the CsCl gradient.
% of empty/incomplete
particles
RfA (mV s mg-1
cm-1
) q (mg/mL) Molar conc. (mM)
Protein 10
Protein 13
Protein 10
Protein 13
Protein 10
Protein 13
0% 305580 235080 0.8 0.1 1.3E-05 2.7E-06
21% 311070 249810 0.8 0.6 1.3E-05 1.3E-05
42% 316560 255330 0.8 1.1 1.3E-05 2.6E-05
62% 321540 249990 0.8 1.7 1.3E-05 3.9E-05
83% 319980 258420 0.8 2.1 1.3E-05 4.7E-05
The correlation between the estimated percentage of incomplete/empty
particles and molar concentration of protein 13 (normalized by dividing it by the
molar concentration of protein 10) is shown in Table 8 and Figure 23.
Table 8. The correlation between the upper band and
the molar concentration ratio of protein 13 and protein 10
% of empty/incomplete particles
Molar conc. ratio of p13/p10
0% 0.2
21% 0.1
42% 2.0
62% 3.0
83% 3.6
Figure 23. Plot of the molar concentration ratio of protein 13 vs protein 10, against the estimated percentage of empty/incomplete particles. The solid line represents the linear regression fit.
y = 4,2454x + 0,185 R² = 0,9955
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
0% 20% 40% 60% 80% 100%
Mo
lar
con
c. r
atio
of
p1
3/p
10
Estimated % of empty or incomplete particles
Purity determination of adenoviruses
42
The percentage of empty/incomplete particles in adenovirus control
samples were determined by calculating the molar concentration ratio of protein
13 vs protein 10 in adenovirus control sample and comparing it to the calibration
curve in Figure 23. The percentages of incomplete particles in the samples of
adenovirus control sample analyzed in this study were between 4.8% and 8.1%.
The percentage of empty/incomplete particles in adenovirus control sample
obtained from the calibration curve were two times lower than if it is calculated
based on AUC result (16%). There were two main reasons for the significant
differences in the results of the two methods. First, the AUC results of adenovirus
particles were determined at difference wavelength. This difference in wavelength
could affect the results due to the absorbance or the light scattering effects of the
adenovirus particles. Second, the AUC results show that there are two different
peaks assigned as empty/incomplete particles. One of the peaks may still contain
DNA whereas the other does not have DNA at all. On the other hand, the
percentages of incomplete/empty particles in adenovirus control sample by RP-
HPLC were determined based on the protein profiles.
In conclusion, the RP-HPLC method with the calculation as described could
be used to calculate the percentage of empty/incomplete particles in adenovirus
samples.
Purity determination of adenoviruses
43
CONCLUSION
Several methods were explored in this study to determine the purity of
adenovirus (quantification of product related-impurities). SEC-HPLC, AEX HPLC and
CE methods did not lead to satisfactory separation of the upper and lower bands of
CsCl gradient. With SEC-HPLC, the retention times of both bands of CsCl gradient
were the same, whereas with AEX-HPLC only slightly different retention times were
observed between the upper and lower bands of CsCl gradients. CE, which is
expected to have high separation efficiency, was also explored to separate the
upper and lower bands of CsCl gradients. Different capillaries and background
electrolyte (BGE) were tried for the experiments, but none of the experiments
yielded satisfactory separation of upper and lower bands. Since none of the
methods could distinguish between upper and lower bands of the CsCl gradient,
we concluded that complete and incomplete/empty adenovirus particles have
similar hydrodynamic volumes and surface charges.
Another technique, RP-HPLC, was explored for distinguishing between the
CsCl fractions. In this method, we obtain the adenovirus particles’ protein profiles
after particle disassembly in an appropriate mobile phase. We observed different
protein profiles for each of the CsCl fractions. Most of the proteins in the profiles
were present in both fractions. We focused our research on protein 13 and protein
10. Protein 13 is associated with adenovirus assembly and encapsidation of the
viral DNA. A large relative concentration of protein 13 in the upper band revealed
that the adenovirus particles in this band lacked or had less DNA. Equal
concentrations of protein 10 in the two CsCl fractions indicated that this protein is
present in the same stoichiometry in complete and incomplete/empty adenovirus
particles.
A calibration curve was used to determine the purity of an adenovirus
control sample (% of product-related impurities). The calibration curve was made
by plotting the molar concentration ratios of protein 13 vs protein 10 against the
percentage of empty/incomplete particles in “calibration samples”. The calibration
Purity determination of adenoviruses
44
samples were prepared by mixing defined volumes of the upper and lower bands
of the CsCl gradient. A good linear correlation was observed between the
estimated percentage of empty/incomplete particles and the molar concentration
ratio of protein 13 vs protein 10. The molar concentration ratio of protein 13 vs
protein 10 of adenovirus control sample indicated that the adenovirus control
sample samples tested contained less than 10% of incomplete particles (estimation
based on the calibration curve described above). The percentage of
empty/incomplete particles in adenovirus control sample determined by AUC
method was significantly different from the RP-HPLC (two times higher). This result
might be caused by the different wavelength and the different measurement
principles that were used for the two determinations.
In summary, RP-HPLC is a promising tool to determine the purity of
adenovirus, in terms of presence of product-related impurities (empty/incomplete
particles). The method can be used to replace the current DCS method that is used
for the same purpose in our laboratories. The percentage of empty/incomplete
particles in adenovirus-based vaccines can be very useful to estimate the vaccine
potency.
Purity determination of adenoviruses
45
FUTURE PERSPECTIVE
It was concluded that RP-HPLC is the method of choice for determining the
percentage of empty/incomplete particles in adenovirus preparations. Currently,
the analysis time of RP-HPLC is more than two hours. The method should be
optimized and validated to obtain a fast and robust analysis. Mass spectrometry
(MS) analysis is needed to confirm the peak assignments in the RP-HPLC
chromatograms. In addition, the correlation between molar concentration of the
protein 13 (normalized to the concentration of protein 10) and the percentage of
empty/incomplete particles needs to be accurately established as the results
reported here are only estimations based on preliminary results (of both AUC and
RP-HPLC).
Although CE and HPSEC were not fit-for-purpose to determine the purity of
adenovirus preparations, these methods can be further developed to determine
virus particles concentrations.
Purity determination of adenoviruses
46
REFERENCES
[1] San Martin C. Latest insights on adenovirus structure and assembly. Viruses;4(5):847-77.
[2] Russell WC. Adenoviruses: update on structure and function. Journal of General Virology 2009;90(1):1-20.
[3] Chawla T, Khanna N, Swaminathan S. Adenovirus-vectored vaccines, 2008. p. 293-307.
[4] Shih S-J, Yagami M, Tseng W-J, Lin A. Validation of a Quantitative Method for Detection of Adenovirus Aggregation. Bioprocessing Journal 2011;9(2):25-33.
[5] Vellekamp G, Porter FW, Sutjipto S, Cutler C, Bondoc L, Liu Y-H, Wylie D, Cannon-Carlson S, Tang JT, Frei A. Empty capsids in column-purified recombinant adenovirus preparations. Human gene therapy 2001;12(15):1923-36.
[6] Shabram P, Vellekamp G, Scandella C. Purification of adenovirus Adenoviral Vectors for Gene Therapy: Academic Press, San Diego, CA, 2002. p. 167-203.
[7] Vellekamp PSG, Scandella C. Purification of Adenovirus. Adenoviral Vectors for Gene Therapy 2002:167-203.
[8] Vegad H. An introduction to particle size characterisation by DCS. [9] Berkowitz SA. Determining the concentration and the absorptivity factor at 260
nm in sodium dodecyl sulfate of the adenovirus reference material using analytical ultracentrifugation. Analytical biochemistry 2008;380(1):152-54.
[10] Berkowitz SA, Philo JS. Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery system) using analytical ultracentrifugation. Analytical biochemistry 2007;362(1):16-37.
[11] Whitfield RJ, Battom SE, Barut M, Gilham DE, Ball PD. Rapid high-performance liquid chromatographic analysis of adenovirus type 5 particles with a prototype anion-exchange analytical monolith column. Journal of Chromatography A 2009;1216(13):2725-29.
[12] Zhang S, Clarke P. CHROMATOGRAPHIC METHODS FOR ASSESSING ADENOVIRUS PURITY: U.S. Patent Application 12/043,557, 2008.
[13] Blanche F, Cameron B, Barbot A, Ferrero L, Guillemin T, Guyot S, Somarriba S, Bisch D. An improved anion-exchange HPLC method for the detection and purification of adenoviral particles. Gene therapy 2000;7(12):1055-62.
[14] Kuhn I, Larsen B, Gross C, Hermiston T. High-performance liquid chromatography method for rapid assessment of viral particle number in crude adenoviral lysates of mixed serotype. Gene therapy 2006;14(2):180-84.
[15] Huyghe BG, Liu X, Sutjipto S, Sugarman BJ, Horn MT, Shepard HM, Scandella CJ, Shabram P. Purification of a type 5 recombinant adenovirus encoding human p53 by column chromatography. Human gene therapy 1995;6(11):1403-16.
[16] Kenndler E, Blaas D. Capillary electrophoresis of macromolecular biological assemblies: bacteria and viruses. TrAC Trends in Analytical Chemistry 2001;20(10):543-51.
[17] Kremser L, Bilek G, Blaas D, Kenndler E. Capillary electrophoresis of viruses, subviral particles and virus complexes. Journal of separation science 2007;30(11):1704-13.
[18] Kremser L, Blaas D, Kenndler E. Capillary electrophoresis of biological particles: viruses, bacteria, and eukaryotic cells. Electrophoresis 2004;25(14):2282-91.
Purity determination of adenoviruses
47
[19] Mann B, Traina JA, Soderblom C, Murakami PK, Lehmberg E, Lee D, Irving J, Nestaas E, Pungor Jr E. Capillary zone electrophoresis of a recombinant adenovirus. Journal of Chromatography A 2000;895(1):329-37.
[20] Desai MJ, Armstrong DW. Separation, identification, and characterization of microorganisms by capillary electrophoresis. Microbiology and molecular biology reviews 2003;67(1):38-51.
[21] Kostal V, Arriaga EA. Recent advances in the analysis of biological particles by capillary electrophoresis. Electrophoresis 2008;29(12):2578-86.
[22] Stewart PL, Burnett RM. Adenovirus structure by X-ray crystallography and electron microscopy The Molecular Repertoire of Adenoviruses I: Springer, 1995. p. 25-38.
[23] Vellinga J, Van der Heijdt S, Hoeben RC. The adenovirus capsid: major progress in minor proteins. Journal of General Virology 2005;86(6):1581-88.
[24] Bondoc Jr LL, Fitzpatrick S. Size distribution analysis of recombinant adenovirus using disc centrifugation. Journal of Industrial Microbiology and Biotechnology 1998;20(6):317-22.
[25] Nyberg-Hoffman C, Aguilar-Cordova E. Instability of adenoviral vectors during transport and its implication for clinical studies. Nature medicine 1999;5(8):955-57.
[26] Stinski MF, Ginsberg HS. Hexon peptides of type 2, 3, and 5 adenoviruses and their relationship to hexon structure. Journal of virology 1975;15(4):898-905.
[27] Hasson TB, Ornelles DA, Shenk T. Adenovirus L1 52-and 55-kilodalton proteins are present within assembling virions and colocalize with nuclear structures distinct from replication centers. Journal of virology 1992;66(10):6133-42.
[28] Klyushnichenko V, Bernier A, Kamen A, Harmsen E. Improved high-performance liquid chromatographic method in the analysis of adenovirus particles. Journal of Chromatography B: Biomedical Sciences and Applications 2001;755(1):27-36.
[29] Yang X, Agarwala S, Ravindran S, Vellekamp G. Determination of particle heterogeneity and stability of recombinant adenovirus by analytical ultracentrifugation in CsCl gradients. Journal of pharmaceutical sciences 2008;97(2):746-63.
[30] Frei M. Centrifugation Separations BioFiles: Sigma-Aldrich, 2011. p. 6-13. [31] Laidlaw I, Steinmetz M. Introduction to differential sedimentation. Analytical
ultracentrifugation: Techniques and methods. Royal Society of Chemistry, Cambridge, UK 2005:270-90.
[32] Europe CI. Analysis of Low Density Particles using Differential Centrifugal Sedimentation. The Netherlands: CPS Instrument Europe.
[33] Peter Lembke BS, S.L. Reus. Liquid Chromatography. In: Helmut Gunzler AW Handbook of Analytical Techniques Verlag GmbH: Wiley-VCH, 2001. p. 262-323.
[34] Transfiguracion J, Jorio H, Meghrous J, Jacob D, Kamen A. High yield purification of functional baculovirus vectors by size exclusion chromatography. Journal of virological methods 2007;142(1):21-28.
[35] Eriksson K, Olmsted EA, Palladino J, Herzer S, Moore P, Davis AR. Rapid Adenovirus Purification using Q Sepharose XL Viral Vectors and Vaccines meeting Lake Tahoe, 2001.
[36] Lehmberg E, Traina JA, Chakel JA, Chang R-J, Parkman M, McCaman MT, Murakami PK, Lahidji V, Nelson JW, Hancock WS. Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome. Journal of Chromatography B: Biomedical Sciences and Applications 1999;732(2):411-23.
Purity determination of adenoviruses
48
[37] Takahashi E, Cohen SL, Tsai PK, Sweeney JA. Quantitation of adenovirus type 5 empty capsids. Analytical biochemistry 2006;349(2):208-17.
[38] Cutler P. Size Exclusion Chromatography. In: Walker JM, Rapley R Molecular Biomethods Handbook: Humana Press, 2008. p. 719-29.
[39] Barth HG, Jackson C, Boyes BE. Size exclusion chromatography. Analytical chemistry 1994;66(12):595R-620R.
[40] California Uo. Chromatographic Columns: University of California, 2010. [41] Hagel L, Jagschies Gn, Sofer GK. Handbook of process chromatography:
development, manufacturing, validation and economics Academic Press, 2007. [42] Aguilar M-I. HPLC of Peptides and Proteins Springer, 2004. [43] Waters. Ion-Exchange Chromatography. New York. [44] Williams A, Frasca V. Ion Exchange Chromatography. Current Protocols in Protein
Science 2001:8.2. 1-8.2. 30. [45] Okafo GN, Roberts JK. 2 Development of achiral separation methods in
pharmaceutical analysis Pharmaceutical Analysis, 2003. p. 31. [46] Xu Y. Tutorial: capillary electrophoresis. The Chemical Educator 1996;1(2):1-14. [47] Kok WT. Capillary electrophoresis: Instrumentation and operation.
Chromatographia 2000;51:S1-S89. [48] Lauer HH, Rozing GP. High Performance Capillary Electrophoresis. Germany:
Agilent Technologies, 2010. p. 1-174. [49] Camilleri P. Capillary electrophoresis: theory and practice CRC PressI Llc, 1998. [50] Altria KD. Fundamentals of capillary electrophoresis theory Capillary
Electrophoresis Guidebook: Springer, 1996. p. 3-13. [51] Li SFY. Capillary electrophoresis: principles, practice and applications Elsevier
Science, 1992. [52] Wikipedia. Capillary Electrophoresis, 2013. [53] Flint SJ. Adenoviruses. eLS 2002. [54] Eberlein GA. Quantitation of proteins using HPLC-detector response rather than
standard curve comparison. Journal of pharmaceutical and biomedical analysis 1995;13(10):1263-71.
[55] Bioinformatics SIo. ExPASy ProtParam.